Human γδT cells occur in the thymus medulla of normal fetuses at 7–8 weeks, and their development process are similar to that of αβT cells. Before gaining autoimmune tolerance, they not only need to undergo functional TCR expression, but also need to undergo negative selection. The point is that some γδT cells have not undergone double positive selection, so that γδT cells have non-limiting MHC when recognizing antigens. Studies have found that the formation of various functions of γδT cells begins in the thymus and matures in the peripheral blood. In the thymus, thymic precursor cells differentiate into γδTCR + thymocytes under the control of the TCR signal pathway. After leaving the thymus, they enter the peripheral blood circulation and become γδT cells in the peripheral blood circulation. So far, the immune function of various γδT cell subtypes has been basically perfected. Subsequently, these cells can differentiate into a single oligoclonal cell subtype under the induction of TCR ligand-related molecules, and further develop under the action of various hormones released by the thymus, and finally have the function of mature immune cells (Cook et al., 2008; Narayan et al., 2012; Shekhar et al., 2012).

The TCRγδ gene contains four groups of genes V (variable region), D (diversity region), J (joining region) and C (constant region). The γ chain gene consists of 10 V gene segments, 2 D gene segments, Two J gene segments are composed of C, while the delta chain gene is composed of only 7 V gene segments, 2 J gene segments and C. The combination of these gene segments and the diversity of junction regions make TCRγδ have the potential for diversity. However, because γδT cell subsets often only use a specific combination of VγVδ and J region sequences, the TCRγδ structure lacks diversity (Chen et al., 2012). The gene recombination and matching of γδT cells are highly coordinated. Vγ9 and Cγ1 are linked and almost all combine with Vδ2 to form TCR dimers. Studies have confirmed that the Vγ9Vδ2 subtype in adult peripheral blood accounts for more than 90% of the total number of γδT cells (Yang et al., 2011). Therefore, the γδT gene lacks diversity with limited gene rearrangement, MHC non-restrictive reaction with antigen (Davey et al., 2018), and similar functions to cells related to natural immunity, so it was initially considered to be an important part of the body’s innate immunity.

γδT cells are not only an important cell group involved in innate immune response, but also a key component of non-specific immune response. The recognition of γδT cells to antigens is not restricted by MHC and can directly recognize antigens. Not only can they recognize complete polypeptides, they can respond to certain MHC-like molecules, and they also show special affinity for heat shock proteins. The recognition of γδT cells to antigens shows certain tissue specificity: γδT cells from the same tissue express the same TCR to recognize antigens of the same nature, while γδT cells from different tissues can express different TCRs to recognize antigens of different properties (Khatri et al., 2010). The antigens recognized by γδT cells currently found mainly include MHC and MHC-like molecules, heat shock proteins (HSP), DNA mismatch repair related proteins (MSH2), phosphorylated antigens, and those presented by CD1a, CD1c, and CD1d in the CD1 family Lipid antigens and so on (Sebestyen et al., 2020). These antigens bind to T cell receptors or NK cell receptors on the surface of γδT cells to cause the activation of γδT. Natural killer receptors (NKR) and Toll-like receptors (TLR) can provide costimulatory signals to participate in the activation process. Parts of the mechanisms of the activation of Vγ9Vδ2+ T cells by phosphoantigens are mediated through the B7 immunoglobulin family-like butyrophilin 2A1 (BTN2A1) and BTN3A1 complexes. Following phosphoantigen binding to the intracellular B30.2 domains of BTN3A1 in tumor cells, BTN3A1 undergoes a conformational change (Sandstrom et al., 2014; Gu et al., 2017) and promotes the interaction between BTN2A1 and BTN3A1 intracellular domains (Rigau et al., 2020). Subsequently, the germline-encoded regions of the TCR Vγ9 chain directly bind to BTN2A1 on tumor cells (Rigau et al., 2020), then leads to Vγ9Vδ2+ T Cell activation. Activated γδT cells exhibit a variety of biological and immunological functions: 1) Non-specific immune response: Without the presentation of APC, it can be activated directly through TCR to recognize multiple antigen components, participate in non-specific immune response and play an important role. 2) Secretion of a variety of cytokines: by secreting cytokines such as TNF-α, IFN-γ, IL-4, IL-10, etc., it can not only directly inhibit tumor growth, but also promote the maturation of dendritic cells and enhance natural killer cell-mediated cytotoxicity (Maniar et al., 2010; McCarthy et al., 2013). 3) Promote target cell apoptosis: destroy the cell structure of target cells by secreting perforin and granzyme B; exert antibody-dependent cell-mediated cytotoxicity through certain membrane surface receptors such as FcγR; through Fas/FasL Pathways, expression-related apoptosis-inducing ligand CD95 ligand and TNF-related apoptosis-inducing ligand (TRAIL), etc. cause programmed apoptosis of target cells (Pennington et al., 2005; Poonia and Pauza, 2012). 4) Antigen presentation: partially activated γδT cells are specialized antigen presenting cells, and their surface highly expresses chemokine receptors CCR7, MHC-Ⅱ molecules, CD80 and CD86, etc., and processes the antigens and cross-presents them to αβT cells thus stimulate a specific immune response (Brandes et al., 2009; Wu et al., 2009). 5) Immune surveillance and immunomodulation: Activated γδ T cells exert immune surveillance through the high expression of CCR7 and CD161 on their surface (Chodaczek et al., 2012); through the production of IL-10, transforming growth factor-β (TGF-β) and other cells factors play an immunomodulatory role (Kühl et al., 2009). 6) Tumor-promoting effect (Figure 1): The tumor-promoting effect of γδT cells is mainly related to the production of IL-17. It not only induces tumor angiogenesis, stimulates tumor cell proliferation, and promotes tumor cell metastasis (Welte and Zhang, 2015; Qian et al., 2017), but also mobilizes pro-inflammatory neutrophils or immunosuppressive myeloid cells. Myeloid-derived suppressor cells inhibit the activation of CD8+T cells through high expression of ARG1 (Romano et al., 2018; Sacchi et al., 2018), and reactive oxygen species (ROS) produced by neutrophils have a certain inhibitory effect on IL-17-producing γδT cells (Wakita et al., 2010; Benevides et al., 2015; Coffelt et al., 2015). Other tumor-promoting effects of γδT cells include inhibiting the maturation of DCs, the senescent DCs can further suppress CD4+T cells and CD8+T cells (Peng et al., 2007; Ye et al., 2013a; Ye et al., 2013b), inhibiting T cell responses by secreting galectin and expressing programmed cell death protein ligand 1 (PDL1); and inducing tumor cell proliferation by expressing IL-22 and biregulin (Daley et al., 2016; Khosravi et al., 2018; Chen et al., 2019; Silva-Santos et al., 2019; Zhang et al., 2020). 7) Anti-tumor effect (Figure 2): Direct anti-tumor effect: activated γδΤ cells can secrete perforin, granzyme B and IFN-γ or express CD95 ligand (CD95L) and TRAIL to directly kill tumor cells (Gao et al., 2003); Indirect anti-tumor effect: Activated γδT cells induce DC maturation and infiltration by the secretion of TNF-α and IFN-γ (Conti et al., 2005; Münz et al., 2005; Nussbaumer et al., 2011); induce robust NK cell-mediated anti-tumor cytotoxicity through CD137 engagement (Maniar et al., 2010); efficiently processed and displayed antigens and provided co-stimulatory signals sufficient for strong induction of naïve αβT cell proliferation and differentiation (Brandes et al., 2005; Khan et al., 2014; Mao et al., 2014); γδT cells can target tumor associated macrophages and MDSCs to improve their anti-tumor ability. The enhanced killing capacity was correlated with the increased CD25 expression and IFN-γ secretion of γδT cells (Li et al., 2022).

1) Gastric cancer: Compared with normal gastric mucosa, the γδT cells in gastric cancer tissue are mainly Vδ1+T cells. The frequency of Vδ1γδT cells in gastric cancer tissue is reduced, the function is impaired, the secretion of IFN-γ and the expression of NKG2D are reduced. Reduced NKG2D expression may be one of the mechanisms of impaired function. The γδT cells infiltrated in gastric cancer tissue are also related to the prognosis of patients and can be used as an independent factor to judge the prognosis of patients (Kuroda et al., 2012; Wang. J et al., 2017; Chen et al., 2017). In vitro, γδT cells induced by pyrophosphate combined with IL-2 exhibited killing activity against gastric cancer cell line SGC-7901. The cytotoxicity of the cell line SGC-7901 was enhanced (Wu et al., 2016). Peripheral-derived γδT cells activated by gastric tumors can not only effectively kill tumor cells, but also induce the activation and proliferation of CD4⁺ and CD8+αβT cells through the antigen-presenting cell properties of Vγ9Vδ2 T cells, and enhance the cytotoxic function of CD8+αβT cells (Mao et al., 2014). In vivo, γδT cells also showed a certain anti-tumor effect, Wada et al. (Wada et al., 2014) used Vδ2+γδT cells induced and cultured in vitro to immunotherapy patients with advanced gastric cancer with malignant ascites. After treatment, the number of tumor cells in the patients’ ascites was significantly reduced and the ascites of some patients were controlled. Postoperative recurrence of gastric cancer is still a common problem, and cellular immunotherapy combined with chemotherapy seems to benefit patients after gastric cancer surgery. Oxaliplatin, a platinum drug for the treatment of gastric cancer, can upregulate the expression of NKG2D in tumor cells, thereby enhancing the sensitivity of tumor cells to kill mediated by γδT cells, NK cells, or cytokine-induced killer (CIK) cells (Gasser et al., 2005). When γδT cells, NK cells, and CIK cells combined with chemotherapy (5-FU and platinum) were used as adjuvant therapy after gastric cancer surgery, the adjuvant therapy showed good resistance compared with chemotherapy alone. Receptivity and safety can improve the quality of life of patients, significantly reduce the risk of recurrence and metastasis of stage II/III gastric cancer, and significantly improve the clinical prognosis of patients with stage II/III gastric cancer (Cui et al., 2015; Wang. Y et al., 2017).

1) Ovarian cancer: A number of studies have shown that, γδT cells have a killing effect on ovarian cancer cells. In vitro, polyclonal γδT cells proliferated and activated by γ-irradiated k562-derived artificial antigen-presenting cells (aAPCs) showed a wide range of anti-tumor activities, and had certain anti-tumor activity against various ovarian cancer cell lines, such as CAOV3, EFO21, UPN251, IGROV1, and OC314. There are obvious inhibitory effects on the transplanted tumor of NSG mice established with ovarian cancer cells CAOV3-effLuc-mKate, and significantly reducing the tumor burden in mice (Deniger et al., 2014). In vitro, Free or liposomal aminobisphosphonic acid salts such as zoledronic acid (ZA) and alendronic acid (AA) can enhance the killing effect of γδT cells on ovarian cancer cell lines SKOV-3 and IGROV1, and patient-derived γδT cells can also kill autologous cells after activation ovarian cancer cells. Intraperitoneal administration of low doses of AA or liposomal AA (L-AA) with γδT cells resulted in modest tumor regression in many SKOV-3-luc xenograft mice, and higher doses of AA or L-AA Intravenous administration with γδT cells resulted in marked and sustained tumor regression in SKOV-3-luc xenograft mice and prolonged survival of the mice. Activation of γδT cells by L-AA was also demonstrated in mice with a more aggressive IGROV-1-luc tumor model. The low maximum tolerated dose of liposomal ZA in SCID mice limits its application in vivo (Parente-Pereira et al., 2014). Foord et al. found that ascites-derived γδT cells had higher killing ability than CD8 + T cells in killing mature ovarian cancer cell line OVCAR-3, produced a higher proportion of IFN-γ, and derived from long-term survivors. γδT cells showed higher killing capacity than deceased patients (Foord et al., 2021). Another study found (Lai et al., 2012) that γδT cells also had an inhibitory effect on cells expressing stem cell markers in ovarian cancer. Researchers co-cultured γδT cells with microspheres with stem cell characteristics induced under certain conditions and found that the microspheres proliferation rate and the expression of stem cell-related genes were significantly reduced, the sensitivity to paclitaxel and cisplatin was increased, and the expression of antigens HLA-DR, B7-1, and B7-2 were significantly increased. It can be seen that γδT cells have a clear inhibitory effect on putative cancer stem cells.

The research on the effect of γδT cells on urinary system tumors is mainly in kidney cancer. Peripheral blood γδT cells of patients with metastatic renal cell carcinoma (MRCC) can selectively act on kidney cancer cells after being activated by nitrogen-containing bisphosphonate and IL-2, but there are no effect on normal kidney cells. The above-mentioned selectivity may be related to the high expression of MICA/B and ULBP in renal cancer cells. γδT cells combine with MICA/B and ULBP through the NKG2D receptor to provide costimulatory signals to enhance the lysis of tumor cells by TCR signals (Viey et al., 2005). Zoledronate combined with IL-15 to induce γδT cells from healthy volunteer PBMCs effectively inhibited the growth of tumors in mice bearing renal cell carcinoma patient-derived xenografts and prolonged the survival time of tumor-bearing mice (Zhang et al., 2021).

The inhibitory effect of γδT cells on renal cell carcinoma has also been studied in a small clinical scale. Kobayashi et al. (Kobayashi et al., 2007) used γδT cells to treat patients with advanced renal cancer after radical nephrectomy. Among the 7 patients, 3 patients had prolonged tumor doubling time and increased the number of γδT cells in peripheral blood. The study also found that the proportion of γδT cells in the peripheral blood of patients was related to the rate of tumor metastasis and the occurrence of tumor-related deaths in patients, which is one of the important factors to improve the prognosis of renal cancer patients. Bennouna et al. (Bennouna et al., 2008) used γδT cells combined with IL-2 to immunotherapy in 10 patients with metastatic renal cell carcinoma. 6 patients were in stable condition and the tumor progression time was prolonged. The immunotherapy of γδT cells may be the gospel for patients with advanced renal cancer.

1) Nasopharyngeal carcinoma: Zheng B et al. (Zheng et al., 2001a) experimentally confirmed that γδT cells obtained by selective expansion of healthy human peripheral blood mononuclear cells in vitro had a certain cytotoxic effect on nasopharyngeal carcinoma cell lines CNE2 and 915, and this effect was related to the number of CD56-γδ T cells. Zheng BJ et al. (Zheng et al., 2001b) found that the use of nasopharyngeal carcinoma cell line CNE2 to establish a nude mouse tumor model, 5 days after CNE2 cell inoculation, tumors were seen subcutaneously in nude mice, and nude mice that were not treated with γδT cells had progressive tumors growth, the average lifespan of mice was 35 ± 3.4 days; CNE2 cells were inoculated with γδT cell treatment on the 10th day, and only a single dose of γδT cell treatment was given to the group. The tumor resumed growth 1 week later, and the average lifespan of mice was 61 ± 15.7 days; Once every other week, the group who was given γδT cell treatment twice resulted in delayed tumor recovery and growth, and the average life span of mice was prolonged by 74 ± 12.9 days. The results of immunohistochemistry showed that the tumor specimens on the second day of γδT cell treatment showed γδT cell accumulation and local necrosis, while on the sixth day, the infiltrating cells in the tumor tissue disappeared and the tumor cell mitosis increased. The above studies suggest that γδT cells exert a certain inhibitory effect on nasopharyngeal carcinoma cell lines both in vivo and in vitro.

The research on the effect of γδT cells on nervous system tumors is more common in neuroblastoma (neurobiastoma, NB). Many studies have confirmed (Schilbach et al., 2000; Chargui et al., 2010) that γδT cells proliferated and activated in vitro are highly cytotoxic to human neuroblastoma cells. They can effectively kill a variety of NB cell lines. γδT cells mainly recognize the target of NB cell line through their TCRγδ, and NKG2D has a weak role in NB cell lysis. This may be related to the lack of MICA on NB cells. These studies show the feasibility of using γδT cells to treat patients with neuroblastoma. Pressey et al. (Pressey et al., 2016) used phosphoantigen combined with IL-2 to perform immunotherapy on patients with refractory neuroblastoma and found that the therapy was well tolerated. The number of γδT cells in the patient increased significantly, and the patient had experienced remission. No toxic side effects of the therapy were found.

γδT cells not only show inhibitory effects on solid tumors, but also have clear killing activity on hematological tumors. γδT cells are effective against many types of acute leukemia cell lines such as Jurkat cell line, THP-1 cell line, HL-60 cell line, chronic myeloid leukemia K562 cell line, multiple myeloma RPMI-8226 cell line and histiocytic lymphoma U-937 cell line, and it has strong killing effect on Jurkat cell line and U-937 cell line (Xiao et al., 2017). D'Asaro et al. (D’Asaro et al., 2010) confirmed that Vγ9Vδ2 T cells could recognize, phagocytose and effectively kill imatinib-sensitive CML cell line K562S and imatinib-resistant CML cell line K562R after pretreatment with zoledronate, KCL22R and LAMA84R, and prolong the survival of Nod/SCID mice bearing the CML cell line MM-1. Almeida et al. (Almeida et al., 2016) found that DOT cells had high cytotoxicity to CLL cell line MEC-1, autologous and allogeneic CLL cells, and had a significant inhibitory effect on NSG mouse xenografts established by MEC-1 cell line. DOT cells selectively target transformed B lymphocytes through their specific TCR mechanism and NKR mechanism, but there was no effect on normal B lymphocytes. DOT cells also showed a certain inhibitory effect on various AML cell lines such as THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1 and KG-1, did not respond to normal leukocytes including CD33⁺ or CD123+ myeloid cells. Adoptive cell therapy with DOT cells reduces AML burden in blood and target organs in various human AML xenograft models and significantly prolongs host survival without significant toxic effects. DOT cells can also target chemoresistance AML cells. These provide a theoretical basis for the application of DOT cells in the treatment of CLL and AML (Lorenzo et al., 2019). γδT cells can directly kill leukemia cells through perforin/granzyme-dependent cytolysis, and they can also act on hematological tumors by secreting cytokines IFN-γ and TNF-α (Gertner-Dardenne et al., 2012; Xiao et al., 2017). Tokuyama et al. (Tokuyama et al., 2008) found that the killing effect of γδT cells on lymphoma was related to the expression level of CD16 molecules on the cell surface. Trastuzumab and tuximab can enhance the killing activity of γδT cells against lymphoma. Wilhelm et al. (Wilhelm et al., 2003) used pamidronate (PAM) combined with IL-2 proliferation and activation of γδT cells in patients with immunotherapy of 19 patients with refractory non-Hodgkin’s lymphoma and multiple myeloma, and found that PAM combined with low. The dose of IL-2 can specifically induce the proliferation of γδT cells, so that the patient’s condition was stable or partially relieved without obvious adverse reactions. These studies have laid the foundation for the clinical application of γδT cells for immunotherapy of hematological tumors.

Jiang Hui et al. (Jiang et al., 2010) found that γδT cells had a strong killing effect on the osteosarcoma cell line HOS, whether in vitro or in tumor-bearing mice. Studies by Lozupone et al. (Lozupone et al., 2004) confirmed that both in vivo activation and adoptive infusion of γδT cells could inhibit the growth of tumors in melanoma-bearing mice and prolong the survival time of tumor-bearing mice. Malignant melanoma cell lines express NKG2D ligand and low expression of MHC-I related antigen A. These molecules bind to the NKG2D receptor of γδT cells to activate the body’s anti-tumor immunity. Compared with healthy people, the peripheral blood γδT cells of melanoma patients increased significantly, and the increased γδT cells were mainly CD3 + CD28-γδT cells, which exerted anti-tumor effects through the expression of a large amount of perforin (Campillo et al., 2007). In B16 melanoma, IFN-γ produced by γδT cells serves as an early and important source of IFN-γ in tumor immune surveillance, plays a critical role in protecting immune responses against tumor development, and modulates tumor antigen-triggered CD4⁺ and CD8⁺ T cells response, thereby enhancing the recognition and potency of cytotoxic T cells against cancer cells (Khan et al., 2014).